Egyptian propolis extract for functionalization of cellulose nanofiber/poly(vinyl alcohol) porous hydrogel along with characterization and biological applications

Bee propolis is one of the most common natural extracts and has gained significant interest in biomedicine due to its high content of phenolic acids and flavonoids, which are responsible for the antioxidant activity of natural products. The present study report that the propolis extract (PE) was produced by ethanol in the surrounding environment. The obtained PE was added at different concentrations to cellulose nanofiber (CNF)/poly(vinyl alcohol) (PVA), and subjected to freezing thawing and freeze drying methods to develop porous bioactive matrices. Scanning electron microscope (SEM) observations displayed that the prepared samples had an interconnected porous structure with pore sizes in the range of 10–100 μm. The high performance liquid chromatography (HPLC) results of PE showed around 18 polyphenol compounds, with the highest amounts of hesperetin (183.7 µg/mL), chlorogenic acid (96.9 µg/mL) and caffeic acid (90.2 µg/mL). The antibacterial activity results indicated that both PE and PE-functionalized hydrogels exhibited a potential antimicrobial effects against Escherichia coli, Salmonella typhimurium, Streptococcus mutans, and Candida albicans. The in vitro test cell culture experiments indicated that the cells on the PE-functionalized hydrogels had the greatest viability, adhesion, and spreading of cells. Altogether, these data highlight the interesting effect of propolis bio-functionalization to enhance the biological features of CNF/PVA hydrogel as a functional matrix for biomedical applications.


Results and discussions
Polyphenol compounds determination of PE. The chemical composition of PE may vary depending on the honey bee's food source. Therefore, it is important to illustrate the chemical composition of PE to emphasise the presence or absence of certain compounds. As shown in Table 1 and Fig. 2, the HPLC detection method was able to identify 18 polyphenol compounds in PE, which play extremely important roles in its biological activity. The polyphenol compounds with highest content illustrated in PE were hesperetin (183.73 µg/mL), chlorogenic acid (96.92 µg/mL), caffeic acid (90.28 µg/mL), daidzein (67.89 µg/mL) and apigenin (66.59 µg/ mL). While the rutin and vanillin had low concentrations of 0.8 and 0.7 µg/mL, respectively. On the other hand, the only catechin not observed in the PE when compared with the standard. Geographical origin and extraction solvent of propolis are the main factors that affect the availability and concentrations of polyphenol compounds, as illustrated in Table 2.
Characterizations of CNF. For the selective oxidation of primary hydroxyl groups in cellulose (Fig. 3A), TEMPO-mediated oxidation is a suitable method for C-6 oxidation to generate a reactive carboxylic groups. Figure 3B shows the FT-IR spectrum of CNF, which exhibits the characteristic peaks for cellulose chains 35 . A new band, however, was seen at 1739 cm −1 that was attributed to the stretching of carbonyl groups (C=O), from the TEMPO oxidation process. The prepared CNF was confirmed by the XRD pattern reported in Fig. 3C. It shows two main diffraction peaks at 16.4° (110) and 23.1° (200) which are characteristic of native cellulose. Also, the crystallinity percentage was estimated according to the Segal method and recorded ∼ 73% 36 . The internal structure of CNF was also examined using transmission electron microscope (TEM) analysis. Figure 3D showed CNF with a micrometric length and a diameter varying from 4 to 20 nm, calculated via image analysis. Moreo-  Microstructure features of the prepared fiber-reinforced hydrogels. Indeed, polymer hydrogels are fabricated by physical or chemical cross-linking, or even both. Physically cross-linked hydrogels are obtained through weak interactions, while chemical covalent bonding is formed when hydrogels are cross-linked chemically. Among them, PVA hydrogels can be simply formed by the freeze-thawing technique. In this study, PEloaded hydrogels based on CNF and PVA were prepared by simple physical crosslinking after successive runs of freezing and thawing followed by freeze drying to produce porous 3D structures. Figure 4A-C illustrates SEM images of the surfaces of the CNF/PVA, CNF/PVA/PE1 and CNF/PVA/PE2 fiber reinforced hydrogels, along with the cross section images for the same hydrogel sample. Figure 4D shows the cross-section images for the same hydrogels. Images of cross section and surface confirm the formation of 3D rough interconnected porous structures with irregular pores varying in size from 10: 100 µm. This microporous structure is very important  www.nature.com/scientificreports/ for cell recruitment because it facilitates the transport of oxygen and nutrients. The introduction of PE into the hydrogel results in a decrease in pore size and surface roughness. This decrease in pore size suggests the presence of PE on the surface of the hydrogel which promotes its fast release when implanted in vivo. This fast release of antibacterial agents such as PE is important to stop the bacterial invasion. Figure 5 illustrates the FT-IR spectra of PE and PE-loaded PVA/CNF hydrogel samples. The IR spectrum of the PE sample, Fig. 5A, shows a broad band centered at 3410 cm −1 , which is typically attributed to the O-H stretching vibration of the phenol compounds 37 . The two bands at 2920 and 2840 cm −1 are related to C-H asymmetric and symmetric stretching of hydrocarbon compounds, respectively 38 . While the bands at 1720 and 1460 cm −1 are assigned to the carbonyl C=O stretching vibration of flavonoid and lipid content. Furthermore, the bands at 1370 and 1271 cm −1 are due to scissor vibrations of C-H groups and C-O-H stretching vibrations, respectively 39 . Finally, the bands at 1165, 1040, and 870 cm −1 are related to the stretching vibration of alkenes C=C bond, the aromatic ether C-O-C bond and the C-H wagging vibration of phenolic compounds 40 .
On the other hand, the FT-IR spectrum of CNF/PVA hydrogel shows a broad band at 3294 cm −1 , which is due to the O-H stretching vibration of hydroxyl group of both PVA and CNF, as shown in Fig. 5B. The bands at 2905, 1708 and 1420 cm −1 belong to C-H stretching, C=O stretching, and CH 2 bending vibrations, respectively. The other bands at 1040 and 870 cm −1 are related to the C-O and CH 2 stretching vibrations 41 . The FT-IR spectra of CNF/PVA/PE1 and CNF/PVA/PE2 fiber reinforced hydrogels are shown in Fig. 5C and D, respectively. The spectra of both samples exhibit peaks related to CNF/PVA hydrogel, with some modifications. For instance, the O-H stretching bands were shifted to 3335 and 3351 cm −1 for the CNF/PVA/PE1 and CNF/PVA/PE2 samples, respectively. Moreover, the C=O stretching bands were observed at 1739 cm −1 for CNF/PVA/PE1 sample and at 1725 cm −1 for the CNF/PVA/PE2 sample. Together, these shifts can be attributed to the interaction between the CNF/PVA polymer matrix and propolis 41 .
Water uptake. The tendency to water uptake or swelling behavior of CNF/PVA, CNF/PVA/PE1, and CNF/ PVA/PE2 fiber reinforced hydrogels is illustrated in Fig. 6. The ratios of water uptake for all samples are high and increased gradually with time. This may refer to the high porous structure of the samples, which allows water to diffuse easily through the hydrogel. As shown in Fig. 6, the samples loaded with PE (CNF/PVA/PE1 and CNF/ PVA/PE2) exhibit lower water uptake ratios than those of neat (CNF/PVA). For example, at a time interval of 12 h, the ratio of water uptake for CNF/PVA is about 907%, while for CNF/PVA/PE1 and CNF/PVA/PE2 are 648% and 590%, respectively. Therefore, the presence of PE in the CNF/PVA/PE1 and CNF/PVA/PE2 fiber rein- Assessment of antimicrobial activity. Propolis is a resinous material extracted from the buds and flowers of plants and used as a protective agent against microbial contamination 42 . Polyphenols are the major chemical components of PE, and they are responsible for its antimicrobial activity 43 . The antimicrobial action of PE and CNF/PVA, CNF/PVA/PE1, and CNF/PVA/PE2 fiber reinforced hydrogels was explored on pathogens for humans using different approaches. The antimicrobial activity of PE is presented in Table 3 and Fig. 7. Although, PE exhibited antimicrobial potential against all of the studied microorganisms, it has no influence on S. typhimurium as reported by Atik et al. 44 . Control CNF/PVA hydrogel has no effect against the studied microorganisms. The results exhibit a significantly higher antimicrobial activity against Gram-positive (S. mutans), with no significant difference between Gram-negative (E. coli) and C. albicans. Moreover, the MIC was also determined through the investigation of antimicrobial activity at different concentrations. The MIC of PE for E. coli was 0.012 mg/mL, 0.05 mg/mL for S. mutans, and 0.025 mg/mL for C. albicans. On the other hand, the CNF/PVA fiber reinforced hydrogels with PE at different concentrations exhibit antimicrobial action against the strains used, with the highest antimicrobial activity being observed in S. typhimurium, followed by S. mutans, and other microbes showing no significant differences. The difference in antimicrobial behavior of the PE could be due to the different cell walls between the Gram-positive and negative bacteria 45 . The activity of PE as antimicrobial agents is related to the high percent of hesperetin (183.7 µg/mL), chlorogenic acid (96.9 µg/mL) and caffeic acid (90.2 µg/mL) as the main polyphenol compounds of PE. As illustrated in Table 1, hesperetin, chlorogenic acid, and caffeic acid were reported as three compounds from PE with high concentrations. Hesperetin was obtained from the enzymatic hydrolysis of hesperidin and evaluated for antimicrobial activity against Gram-positive and Gram-negative bacteria, the results revealed that hesperetin more effectively inhibited the model microbes than hesperidin and hesperidin glucoside 46 . Yen et al. 47 . found that chlorogenic acid was one of the main components of the water extract of green coffee beans under high pressure and demonstrated antibacterial activity against both Gram-positive (Staphylococcus aureus and Listeria innocua) and Gram-negative (Escherichia coli and Salmonella enterica). Caffeic acid and its combination with antibiotic phytochemicals were evaluated against Staphylococcus aureus, and the results showed diverse effects against Staphylococcus aureus, with the MIC varying from 256 to 1024 µg/mL 48 . Most of the tested polyphenol compounds were not effective against the model microbes used when tested as a single compound, but when combined with other compounds, the activity of the antimicrobials increased. According to Ahmed et al. 49 gallic acid at 200 and 400 µg/mL was effective against S. aureus and S. pyogenes, but not against Gram-negative bacteria. On the other hand, the antisolvent extracted from olive mill wastewater combined with gallic acid was tested using low minimal inhibitory concentrations,  www.nature.com/scientificreports/ which revealed that 50/100-100/100 µg/mL caused complete growth inhibition of all the bacteria used, owing to the synergistic effects of phenolic compounds against the model bacteria used. Other study report that polyphenol compounds extracted from grape pomace combined with representatives of different classes of antibiotics such as β-lactam, quinolone, fluoroquinolone, tetracycline, and amphenicol act in synergy in all S. aureus and E. coli strains tested with fractional inhibitory concentration index (FICI) values varying from 0.031 to 0.155. The MIC was reduced 4 to 75 times due to the synergistic effects of phenolic compounds with different antibiotics 50 .
Previous studies promoted the composite membrane by propolis to improve their biological assessments, for example polylactic acid/essential oil 51 , poly-ε-caprolactone 52 , bacterial cellulose/ZnO-NPs 53 , chitosan/Ag-NPs 54 , and polyurethane/ nanolignin 55 . We can summarize the main bioactive polyphenol compounds from PE as having antimicrobial activity in Table 4.
Cell viability. HFB4 cells were incubated with PE for 24 h at 37 °C in medium to develop a complete monolayer sheet. Different concentrations of PEs were applied up to concentrations of 1000 µg/mL. We observed that PE at concentrations greater than 125 µg/mL significantly reduced cell viability according to MTT (Fig. 8).
Concentrations up to 125 µg/mL did not affect cell viability. Furthermore, light microscopy was used to confirm the cellular proliferation upon treatment of HFB4 cells with different doses of PEs. A decrease of cell number upon increasing PE concentration was clearly seen as compared to the control (Fig. 8). These results indicate that concentrations of PEs beyond the limit of 125 µg/mL may induce harmful effects in HFB4 cells. Previous studies demonstrated that supplementation with natural extracts may increase reactive species production and oxidative stress in vitro and in vivo tests, leading to oxidative damage and cell death 73,74 .  SEM images of the spreading and adhesion of HFB4 cells treated with CNF/PVA, CNF/PVA/PE1, and CNF/ PVA/PE2 fiber reinforced hydrogels are shown in Fig. 9. As seen, all seeded cells have a rounded shape, which may be caused by the dehydration during the fixation process. The spreading of cells on samples loaded with propolis (CNF/PVA/PE1 and CNF/PVA/PE2) is higher compared to a control sample without PE (CNF/PVA). Also, the spreading and attachment of cells in the sample with a higher content of PE (CNF/PVA/PE2) is greater than in the sample with a lower content of PE (CNF/PVA/PE1). Therefore, the presence of propolis enhances cell growth and adhesion. Overall, the in vitro tests performed in this work illustrate the importance of the prepared propolisreinforced hydrogel as a cytocompatible material promoting cell adhesion and spreading in a 3D environment.

In vitro anti-inflammatory tests (hemolysis inhibition). The stabilization of erythrocyte membranes
is an established assay to investigate the anti-inflammatory effect of implanted biomaterials. Inflammation occurs due to release of different enzymes from decomposed lysosomal vesicles 75 . Therefore, stabilization of erythrocyte membrane prevents leakage of these enzymes, as well as incidence of inflammation 75 . Hemolysis assay is very critical assay especially for materials that will be in contact with the blood. Table 5 explains the results of hemolysis inhibition for PE, samples of CNF/PVA hydrogel and those loaded with propolis (CNF/PVA/PE1 and CNF/PVA/PE2). The indomethacin drug was used as the slandered (positive control) and it has the highest protection (100 ± 2.362%) at a concentration of 200 µg/mL against hypotonicity induced lysis of RBCs. As shown in Table 2, all samples protect human erythrocytes in a concentration dependent manner. The protective effect of CNF/PVA/PE2 sample and PE is almost the same at different concentrations. The lowest protective effect is recorded for the CNF/PVA sample (without PE). From these findings, it's clear that, the PE loaded hydrogels are promising as anti-inflammatory agents.

Conclusion
Propolis is a natural byproduct with high value for diverse biological applications. The HPLC analyses of PE revealed about 18 polyphenol compounds, with the highest concentration being hesperetin, followed by chlorogenic acid and caffeic acid. CNF was prepared by oxidation-defibrillation of cellulose obtained from bleached sugarcane bagasse, and used to prepare sustainable and porous CNF/PVA loaded with various PE concentrations. The prepared scaffolds exhibited a high porous structure with pore sizes up to 100 µm. The obtained PE-loaded CNF/PVA hydrogels have a highly antimicrobial activity against E. coli, S. mutans bacteria and then C. albicans. The cellular responses also showed that the cells on the PE-functionalized CNF/PVA hydrogels revealed an optimal microenvironment for the attachment and proliferation of HFB4 cells. Remarkably, our results suggested that PE-functionalization of CNF/PVA not only enhanced the antimicrobial features of PE-loaded CNF/ PVA hydrogels, but also resulted in significant anti-inflammation potential. Thus, in vivo experiments are highly recommended prior to clinical application to underline the potency of PE-loaded CNF/PVA hydrogels in wound healing. Preparation of PE. PE was collected according to Bozkuş et al. 31 with little modifications. In brief, about 10 g of raw material was dissolved in 100 mL of 70% ethanol and then placed in an incubator at 37 °C for 14 days in a dark place. After filtration using Whatman filter paper (No. 1), the mixture was centrifuged at 5000 rpm for   www.nature.com/scientificreports/ Preparation of CNF. Bleached bagasse was used for the preparation of TEMPO-oxidized CNF by using the method described by Salama et al. 35 . In brief, 20 g of bleached bagasse pulp were dispersed in distilled water with TEMPO (0.8 g) and sodium bromide (8 g). Following this, 300 mL of sodium hypochlorite solution (15%) was continuously added and the pH was adjusted to 10 using NaOH (3 mol/L) solution. At the end of the reaction, the pH was reduced to 7.0 and the product was centrifuged several times at 10,000 rpm. Dialysis using deionized water was performed to purify the product.

Preparation of CNF/PVA hydrogel reinforced by PE.
The CNF/PVA porous hydrogel was prepared using the freeze-thawing and freeze drying methods according to Müller et al. 77 . The polymeric weight ratio was adjusted to 50:50 (CNF:PVA). In brief, 30 mL of PVA solution with a concentration of 10 wt% was prepared in distilled water using magnetic stirring at 95 °C. After 4 h, 100 mL of CNF solution (3 wt%) was added to PVA solution and underwent mechanical agitation for 10 min at 5000 rpm. Then, the CNF/PVA mixture was poured into a 10 cm round mould and freezen at − 40 °C for 3 h, followed by thawing at room temperature for 3 h to allow PVA crosslinking. The obtained hydrogels were subjected to four freeze-thawing cycles and subsequently freeze-dried at − 80 °C. To produce PE loaded CNF/PVA hydrogels, different proportions of the PE (0.1 and 0.2 g) were dissolved in 2 mL of ethanol and then added to 22 mL of the CNF/PVA suspension solution mixture. The mixtures were kept on a magnetic stirrer at 40 °C for 1 h to assure complete dissolution of PE. The solutions were homogenized using mechanical agitation for 10 min at 5000 rpm before being poured into round mold and subjected to freeze-thawing cycle and finally lyophilized at − 80 °C, as mentioned above. The prepared samples were named CNF/PVA for control (without PE), CNF/PVA/PE1 for sample with 0.1 g PE and CNF/PVA/PE2 for sample with 0.2 g PE.  www.nature.com/scientificreports/ loaded with different concentrations of propolis. The fiber reinforced hydrogel without propolis was used as a control.
Incubation conditions and assay. All the plates were left at 4 °C for 120 min. to complete the diffusion of the tested sample as well as inhibit the model microbes, subsequently incubated at 37 °C for 1 day, where the antibacterial activity was assessed by measuring the developed inhibition-zone diameter (including the well or membrane disk) after the incubation period. To ensure the aseptic conditions, all membranes were sterilized for 30 min under UV light prior to application. All experiments were conducted in triplicate, and the mean results were represented.
Cytotoxicity assay. Cytotoxicity assay of CNF/PVA, CNF/PVA/PE1, and CNF/PVA/PE2 fiber reinforced hydrogels and also PE against HFB4 cells was performed using the MTT (3-4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay 82,83 . The prepared hydrogels were rinsed with media three times and then plated in the bottom of a 24 well tissue culture plate. The 24 well plate was inoculated with 1 × 10 5 cells/ mL (1000 µL/well) and incubated at 37 °C for 24 h to develop a complete monolayer sheet. Cells were observed for sheet formation and cytotoxicity effects. The HFB4 cells were checked for any physical signs of toxicity, e.g., complete or partial loss of the monolayer, rounding, shrinkage, or cell granulation. MTT solution was prepared (5 mg/mL in PBS) (BioBasic Canada Inc). 100 µL MTT solution was appended to each well. Put on a shaking table, 150 rpm for 5 min, to thoroughly blend the MTT into the media. Incubate (37 °C, 5% CO 2 ) for 4 h to allow the MTT to be metabolized. After that, 200 μL of DMSO was mixed with the culture cells to solubilize the formazan crystals, which were obtained via the reduction of MTT in living cells. Subsequently, the mean absorbance of each well at 570 nm was calculated using a spectrophotometer. Three parallel experiments were performed. The absorbance should be directly correlated with cell quantity 82,83 . For PE, the 24 well tissue culture plate was inoculated with 1 × 10 5 cells/mL (100 µL/well) and incubated at 37 °C for 24 h to develop a complete monolayer sheet. Growth medium was decanted from 24 well microtiter plates after a confluent sheet of cells was formed. Then the cell monolayer was washed twice with wash media. A twofold dilution of the tested sample was made in RPMI medium with 2% serum (maintenance medium). 0.1 mL of each dilution was tested in different wells, leaving 3 wells as controls, receiving only maintenance medium. Then the same steps were performed as mentioned above.
Cell adhesion. HFB4 cells were grown in 24-well plates containing CNF/PVA, CNF/PVA/PE1, and CNF/PVA/ PE2 fiber reinforced hydrogels 84 . After 24 h incubation, the cells were fixed with 5% (V/V) glutaraldehyde and then dehydrated gradually with serial solutions of ethanol (70-100% for 20 min each). After that, the samples sputtered with gold for SEM observations.
Anti-inflammatory assay. The preparation of erythrocyte suspension was done according to Anosike et al. 75 . In brief, about 3 mL of fresh whole blood gathered from healthy volunteers was placed in heparinized tubes and then centrifuged at 3000 rpm for 10 min. A volume of normal saline equal to that of the supernatant was utilized to dissolve the red blood pellets. The volume of the obtained dissolved red blood pellets was measured and returned as a 40% v/v suspension with an isotonic buffer solution (10 mM sodium phosphate buffer, pH 7.4). The buffer solution was formed from 0.2 g of NaH 2 PO 4 , 1.15 g of Na 2 HPO 4 and 9 g of NaCl in 1 L of distilled water. The returned red blood cells (resuspended supernatant) were used as such.
Hypotonicity induced hemolysis. Equal weights of CNF/PVA, CNF/PVA/PE1, and CNF/PVA/PE2 fiber reinforced hydrogels were ground carefully and suspended in distilled water (hypotonic solution). 5 mL of the hypotonic solution of graded doses of the samples (100, 200, 400, 600, 800, and 1000 µg/mL) were placed into duplicate pairs (per dose) of the centrifuge tubes. 5 mL of isotonic solution of graded doses of the samples (100-1000 µg/mL) were also placed into duplicate pairs (per dose) of the centrifuge tubes. The same previous steps were occurred for PE resin, but without grinding. A tube containing 5 mL of the vehicle (distilled water) and another tube containing 5 mL of 200 µg/mL of indomethacin were used as the control tubes, respectively. A suspension (0.1 mL) of erythrocytes was added to each of the tubes and mixed gently. The mixtures were incubated for 1 h at room temperature (37 °C), and afterwards, they were centrifuged for 3 min at 1300 rpm. The absorbance (Ab) of hemoglobin within the supernatant was estimated at 540 nm using a Spectronic (Milton Roy) spectrophotometer. To calculate the hemolysis percentage, the hemolysis formed in the presence of distilled water was assumed to be 100%. The percent inhibition of hemolysis by the extract was calculated thus: where Ab 1 = absorbance of test sample in isotonic solution, Ab 2 = absorbance of test sample in hypotonic solution, Ab 3 = absorbance of control sample in hypotonic solution 75,85 . Statistical analysis. All experiments were done in triplicate, and the results were presented as mean ± standard deviation. Statistical analyses were performed with the one-way ANOVA test, by using Sigma Stat 3.5 software (Dundas Software Ltd, Toronto; Canada). P values ≤ 0.05 were considered statistically significant.

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.